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FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression

View Article: PubMed Central - PubMed

ABSTRACT

Hormonal regulation of gene expression by androgen receptor (AR) is tightly controlled by many transcriptional cofactors, including pioneer factors FOXA1 and GATA2, which, however, exhibit distinct expression patterns and functional roles in prostate cancer. Here, we examined how FOXA1, GATA2, and AR crosstalk and regulate hormone-dependent gene expression in prostate cancer cells. ChIP-seq analysis revealed that FOXA1 reprograms both AR and GATA2 cistrome by preferably recruiting them to FKHD-containing genomic sites. By contrast, GATA2 is unable to shift AR or FOXA1 to GATA motifs. Rather, GATA2 co-occupancy enhances AR and FOXA1 binding to nearby ARE and FKHD sites, respectively. Similarly, AR increases, but not re-programs, GATA2 and FOXA1 cistromes. Concordantly, GATA2 and AR strongly enhance the transcriptional program of each other, whereas FOXA1 regulates GATA2- and AR-mediated gene expression in a context-dependent manner due to its reprogramming effects. Taken together, our data delineated for the first time the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggest that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer.

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Context-dependent roles of FOXA1 in regulating GATA2 programA–B. GATA2 positively regulate FOXA1-targe genes under both androgen-depleted and –replenished conditions. FOXA1-induced genes in the presence (FBS) and absence (FCS) of androgen was derived from microarray data of control and FOXA1-knockdown cells. GSEA of these gene sets was performed in microarray data of control and GATA2-knockdown cells grown under the same androgen environment.C–D. GATA2-induced genes are significantly enriched for up-regulation upon FOXA1 knockdown in androgen-depleted cells, whereas in the presence of androgen GATA2-induced genes tend to become down-regulated following FOXA1 depletion. GATA2-induced gene sets in the presence and absence of androgen were obtained from corresponding microarray data and subjected to GSEA in microarray data of control and FOXA1-knockdown cells.E. QRT-PCR validating that FOXA1 knockdown increased GATA2/AR-induced gene expression. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.F. Genome Browser showing increased GATA2 binding at CXCL10 enhancer and GBP2 promoter following FOXA1 depletion in LNCaP cells.G. QRT-PCR showing decreased CXCL10 and GBP2 levels following GATA2 knockdown. Androgen-deprived and –stimulated LNCaP cells were subjected to control or GATA2 knockdown through RNA interference and then qRT-PCR analysis.H. FOXA1 depletion led to increased expression of GATA2-induced genes. QRT-PCR was performed in control and FOXA1-knockdown LNCaP cells grown in the presence or absence of androgen.
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Figure 6: Context-dependent roles of FOXA1 in regulating GATA2 programA–B. GATA2 positively regulate FOXA1-targe genes under both androgen-depleted and –replenished conditions. FOXA1-induced genes in the presence (FBS) and absence (FCS) of androgen was derived from microarray data of control and FOXA1-knockdown cells. GSEA of these gene sets was performed in microarray data of control and GATA2-knockdown cells grown under the same androgen environment.C–D. GATA2-induced genes are significantly enriched for up-regulation upon FOXA1 knockdown in androgen-depleted cells, whereas in the presence of androgen GATA2-induced genes tend to become down-regulated following FOXA1 depletion. GATA2-induced gene sets in the presence and absence of androgen were obtained from corresponding microarray data and subjected to GSEA in microarray data of control and FOXA1-knockdown cells.E. QRT-PCR validating that FOXA1 knockdown increased GATA2/AR-induced gene expression. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.F. Genome Browser showing increased GATA2 binding at CXCL10 enhancer and GBP2 promoter following FOXA1 depletion in LNCaP cells.G. QRT-PCR showing decreased CXCL10 and GBP2 levels following GATA2 knockdown. Androgen-deprived and –stimulated LNCaP cells were subjected to control or GATA2 knockdown through RNA interference and then qRT-PCR analysis.H. FOXA1 depletion led to increased expression of GATA2-induced genes. QRT-PCR was performed in control and FOXA1-knockdown LNCaP cells grown in the presence or absence of androgen.

Mentions: Next, we examined the downstream transcriptional effects of GATA2 depletion on FOXA1 and AR targets, and vice versa, in order to obtain a better understanding of the regulatory hierarchy between these transcription factors. We first examined FOXA1- or GATA2-regulated genes in androgen-depleted cells. GSEA showed that FOXA1-induced genes are strongly enriched for down-regulation by GATA2 knockdown, supporting GATA2 as a collaborating transcription factor (Figure 6A). Consistent with our ChIP-seq data, GATA2 also positively regulates, although to somewhat lesser extent, FOXA1-induced genes in the presence of androgen (Figure 6B). Therefore, regardless of the androgen environment, GATA2 expression enhances FOXA1 transcriptional activities.


FOXA1 acts upstream of GATA2 and AR in hormonal regulation of gene expression
Context-dependent roles of FOXA1 in regulating GATA2 programA–B. GATA2 positively regulate FOXA1-targe genes under both androgen-depleted and –replenished conditions. FOXA1-induced genes in the presence (FBS) and absence (FCS) of androgen was derived from microarray data of control and FOXA1-knockdown cells. GSEA of these gene sets was performed in microarray data of control and GATA2-knockdown cells grown under the same androgen environment.C–D. GATA2-induced genes are significantly enriched for up-regulation upon FOXA1 knockdown in androgen-depleted cells, whereas in the presence of androgen GATA2-induced genes tend to become down-regulated following FOXA1 depletion. GATA2-induced gene sets in the presence and absence of androgen were obtained from corresponding microarray data and subjected to GSEA in microarray data of control and FOXA1-knockdown cells.E. QRT-PCR validating that FOXA1 knockdown increased GATA2/AR-induced gene expression. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.F. Genome Browser showing increased GATA2 binding at CXCL10 enhancer and GBP2 promoter following FOXA1 depletion in LNCaP cells.G. QRT-PCR showing decreased CXCL10 and GBP2 levels following GATA2 knockdown. Androgen-deprived and –stimulated LNCaP cells were subjected to control or GATA2 knockdown through RNA interference and then qRT-PCR analysis.H. FOXA1 depletion led to increased expression of GATA2-induced genes. QRT-PCR was performed in control and FOXA1-knockdown LNCaP cells grown in the presence or absence of androgen.
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Figure 6: Context-dependent roles of FOXA1 in regulating GATA2 programA–B. GATA2 positively regulate FOXA1-targe genes under both androgen-depleted and –replenished conditions. FOXA1-induced genes in the presence (FBS) and absence (FCS) of androgen was derived from microarray data of control and FOXA1-knockdown cells. GSEA of these gene sets was performed in microarray data of control and GATA2-knockdown cells grown under the same androgen environment.C–D. GATA2-induced genes are significantly enriched for up-regulation upon FOXA1 knockdown in androgen-depleted cells, whereas in the presence of androgen GATA2-induced genes tend to become down-regulated following FOXA1 depletion. GATA2-induced gene sets in the presence and absence of androgen were obtained from corresponding microarray data and subjected to GSEA in microarray data of control and FOXA1-knockdown cells.E. QRT-PCR validating that FOXA1 knockdown increased GATA2/AR-induced gene expression. Data shown are mean ± SEM in triplicate qPCR and is a representative of at least two independent experiments.F. Genome Browser showing increased GATA2 binding at CXCL10 enhancer and GBP2 promoter following FOXA1 depletion in LNCaP cells.G. QRT-PCR showing decreased CXCL10 and GBP2 levels following GATA2 knockdown. Androgen-deprived and –stimulated LNCaP cells were subjected to control or GATA2 knockdown through RNA interference and then qRT-PCR analysis.H. FOXA1 depletion led to increased expression of GATA2-induced genes. QRT-PCR was performed in control and FOXA1-knockdown LNCaP cells grown in the presence or absence of androgen.
Mentions: Next, we examined the downstream transcriptional effects of GATA2 depletion on FOXA1 and AR targets, and vice versa, in order to obtain a better understanding of the regulatory hierarchy between these transcription factors. We first examined FOXA1- or GATA2-regulated genes in androgen-depleted cells. GSEA showed that FOXA1-induced genes are strongly enriched for down-regulation by GATA2 knockdown, supporting GATA2 as a collaborating transcription factor (Figure 6A). Consistent with our ChIP-seq data, GATA2 also positively regulates, although to somewhat lesser extent, FOXA1-induced genes in the presence of androgen (Figure 6B). Therefore, regardless of the androgen environment, GATA2 expression enhances FOXA1 transcriptional activities.

View Article: PubMed Central - PubMed

ABSTRACT

Hormonal regulation of gene expression by androgen receptor (AR) is tightly controlled by many transcriptional cofactors, including pioneer factors FOXA1 and GATA2, which, however, exhibit distinct expression patterns and functional roles in prostate cancer. Here, we examined how FOXA1, GATA2, and AR crosstalk and regulate hormone-dependent gene expression in prostate cancer cells. ChIP-seq analysis revealed that FOXA1 reprograms both AR and GATA2 cistrome by preferably recruiting them to FKHD-containing genomic sites. By contrast, GATA2 is unable to shift AR or FOXA1 to GATA motifs. Rather, GATA2 co-occupancy enhances AR and FOXA1 binding to nearby ARE and FKHD sites, respectively. Similarly, AR increases, but not re-programs, GATA2 and FOXA1 cistromes. Concordantly, GATA2 and AR strongly enhance the transcriptional program of each other, whereas FOXA1 regulates GATA2- and AR-mediated gene expression in a context-dependent manner due to its reprogramming effects. Taken together, our data delineated for the first time the distinct mechanisms by which GATA2 and FOXA1 regulate AR cistrome and suggest that FOXA1 acts upstream of GATA2 and AR in determining hormone-dependent gene expression in prostate cancer.

No MeSH data available.


Related in: MedlinePlus